St. John's wort extracts and some of their constituents potently inhibit ultimate carcinogen formation from benzo[a]pyrene-7,8-dihydrodiol by human CYP1A1.

Commercially available St. John's wort (Hypericum perforatum) preparations and some of their main constituents (hypericin, pseudohypericin, hyperforin, rutin, and quercetin) were examined for their potential to inhibit carcinogen activation by human cytochrome P450 1A1 (CYP1A1). We used a reconstituted system consisting of purified human CYP1A1, purified human NADPH-cytochrome P450 reductase, and dilaurylphosphatidylcholine as lipid component. St. John's wort extracts potently inhibited CYP1A1-catalyzed (+/-)-trans-7,8-dihydro-7,8-dihydroxy-benzo(a)pyrene (7,8-diol-B[a]P) epoxidation, the terminal reaction leading to the ultimate carcinogenic product (+/-)-B[a]P-r-7,t-8-dihydrodiol-t-9,10-epoxide (diolepoxide 2). All constituents, except rutin, were shown to possess strong inhibitory potencies toward diolepoxide 2 formation from 7,8-diol-B[a]P, with IC(50) values of 0.5 microM (hypericin), 1.2 microM (hyperforin), 1.5 microM (quercetin), and 8 microM (pseudohypericin), respectively. Preincubation experiments revealed that their action was not mechanism based. Inhibition kinetics studies showed the anthrodianthrone compound hypericin to be a noncompetitive inhibitor, with a K(i) value of 0.6 microM, and the phloroglucinol hyperforin to be a competitive inhibitor, with a K(i) value of 1.1 microM. When the effects on NADPH-P450 reductase activity were investigated, all constituents of St. John's wort studied turned out to be rather ineffective inhibitors; quercetin was the only exception, with an IC(50) value of approximately 20 microM. These in vitro data indicate that St. John's wort extracts and some of their constituents potently inhibit the major human procarcinogen-activating enzyme CYP1A1.

Inhibition of clastogenicity of benzo[a]pyrene and of its trans-7,8-dihydrodiol in mice in vivo by fruits, vegetables, and flavonoids.

In the in vivo mouse bone marrow micronucleus assay, homogenates of spinach, artichoke, peaches, and blue grapes as well as commercial concentrates of these vegetables and fruits reduced induction of micronuclei by benzo[a]pyrene (BaP) by 43-50%. Concentrates of strawberries (31% reduction) and of cauliflower (20% reduction) were less potent. Inhibition of genotoxicity by spinach and peaches was not caused by any delay in maturation of micronucleated erythrocytes as shown by experiments with sampling times of 24, 48, and 72 h after dosing of BaP. Pre-treatment of the mice with spinach 48, 24, and 12h before application of BaP resulted in a 44% reduction of micronuclei while peaches generated only a marginal effect. A post-treatment procedure administering spinach or peaches 6h after dosing of BaP did not indicate any protective effects. When trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BaP-7,8-OH) was applied for induction of micronuclei spinach and peaches reduced the number of micronuclei by 55 and 48%, respectively. Pre-treatment of mice with spinach 96, 72, and 60 h before sacrifice caused a decline of hepatic 7-ethoxyresorufin-O-dealkylase (EROD) and of 7-pentoxyresorufin-O-dealkylase (PROD) activities by factors of 2.2 and 1.4, respectively. However, statistical significance was not reached. On the other hand, peaches had no influence on hepatic EROD or PROD activities. The flavonoids quercetin and its glucoside isoquercitrin, administered orally in doses of 0.03 mmol/kg body weight simultaneously with intraperitoneally given BaP, reduced the number of micronuclei in polychromatic erythrocytes of the bone marrow of mice by 73 and 33%. Ten-fold higher concentrations, however, reversed the effects with a particular strong increase observed with isoquercitrin (+109%; quercetin: +16%).

Curcumin activates the aryl hydrocarbon receptor yet significantly inhibits (-)-benzo(a)pyrene-7R-trans-7,8-dihydrodiol bioactivation in oral squamous cell carcinoma cells and oral mucosa.

The development of oral squamous cell carcinoma (SCC) shows a positive correlation with the carcinogen exposure that occurs during tobacco and alcohol use. The purpose of this study was to investigate whether the naturally occurring chemopreventive agent, curcumin, modulates expression and function of carcinogen- metabolizing enzymes in human keratinocytes isolated from oral SCC tumors. Dose-response studies demonstrated that curcumin concentrations of >or=25 micro M were cytotoxic for oral SCC cells. Curcumin increased both expression (reverse transcription-PCR analyses) and function (high-performance liquid chromatography determination of ethoxyresorufin metabolism) of cytochrome P-450 (CYP) 1A1 and/or CYP1B1. The aryl hydrocarbon receptor (AhR), which up-regulates a battery of genes associated with carcinogen metabolism, is activated by polycyclic aromatic hydrocarbons such as the tobacco-associated carcinogen benzo(a)pyrene. Electromobility shift assays demonstrated that similar to the established AhR ligand 2,3,7,8,-tetrachlorodibenzo-p-dioxin, curcumin inclusion resulted in AhR nuclear translocation and formation of the transcriptionally active AhR-aryl hydrocarbon receptor nuclear translocator complex. Cellular capacity to bioactivate the tobacco-associated carcinogen (-)-benzo(a)pyrene-7R-trans-7,8-dihydrodiodiol was determined by evaluating conversion of the carcinogenic metabolite diol epoxide to stable tetrols via high-performance liquid chromatography. Results of our metabolism studies showed that curcumin significantly inhibited CYP1A1-mediated benzo(a)pyrene diol bioactivation in both oral SCC cells and intact oral mucosa. Because CYP1A1 is one of the primary carcinogen-activating enzymes in oral mucosa, the use of curcumin as an oral cavity chemopreventive agent could have significant clinical impact via its ability to inhibit carcinogen bioactivation.

Factors influencing elevation of intracellular Ca2+ in the MCF-10A human mammary epithelial cell line by carcinogenic polycyclic aromatic hydrocarbons.

Carcinogenic polycyclic aromatic hydrocarbons and a halogenated aromatic hydrocarbon, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), were evaluated for their effects on intracellular Ca2+ in the human mammary epithelial cell line MCF-10A. After two 18-h incubations with MCF-10A cells, benzo[a]pyrene (BaP; 1, 3, and 10 microM) produced a dose-dependent increase in intracellular Ca2+. 7,12-Dimethylbenz[a]anthracene increased Ca2+ at 10 microM, whereas 3-methylcholanthrene and TCDD did not. The Ca2+-elevating effect of BaP appeared to be dependent on the influx of extracellular Ca2+, as addition of the Ca2+ chelator EGTA to the extracellular medium prevented the increase in Ca2+. MCF-10A cells were found by polymerase chain reaction to express cytochrome P4501A and P4501B isozymes as well as the aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator mRNAs associated with cytochrome P450 induction. Certain cytochrome P450-derived metabolites, including benzo[a]pyrene-7,8-diol (BP-diol) and benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE), were more effective in increasing Ca2+ than was BaP. The Ca2+-elevating effect of BP-diol was prevented by alpha-naphthoflavone, a cytochrome P4501A and P4501B inhibitor, but not by the antioxidant N-acetylcysteine. These results suggest that cytochrome P450-dependent formation of BPDE from BP-diol is a major mechanism required for elevation of Ca2+ in MCF-10A cells.

Piperine impairs cytochrome P4501A1 activity by direct interaction with the enzyme and not by down regulation of CYP1A1 gene expression in the rat hepatoma 5L cell line.

Modulation of benzo(a)pyrene (BP) metabolism and regulation of CYP1A1 gene expression by piperine in 5L cells in culture was studied. Treatment of cultures with 60 microM piperine for different time periods inhibited metabolism of BP by 50% within 4-8 h. Piperine uptake in 5L cells attained saturation plateau at 8 h and this related with the maximal impairment of BP metabolism. Exposure of cell cultures to piperine for 24 h indicated activation of CYP1A1 gene transcription. There was a 10-fold increase in CYP1A1mRNA and an approximately 7-fold increase in arylhydrocarbon hydroxylase (AHH) activity, while treatment with benzanthacene (BA) increased CYP1A1mRNA by 86- and AHH by 56-fold. Combined treatment with BA plus piperine further increased CYP1A1mRNA contents by about 25%. The BA-inducible AHH activity, however, registered a decrease of about 45%. Piperine neither destroyed CYP1A1-protein nor affected the total cellular protein contents. Exposure of cultures to 0.01 to 3.0 microM trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene for 24 h reduced maximum cellular growth by about 50%. Piperine at 60 microM offered full protection against the diol cytotoxicity. The results, suggest that piperine mediated inhibition of the AHH activity and consequent suppression of the procarcinogen activation is the result of direct interaction of piperine with CYP1A1-protein and not because of down regulation of the CYP1A1 gene expression.

In vitro inhibition of the metabolism and mutagenicity of benzo(a)pyrene and benzo(a)pyrene-7,8-dihydrodiol by naphthazarin and other naphthol derivatives.

Among naphthol derivatives tested in the Ames assay, 5,8-dihydroxy-1,4-naphthoquinone or naphthazarin was found to be the most effective inhibitor of benzo(a)pyrene mutagenicity. The inhibitory activity is due in part to the redox cycling of naphthazarin with the concommitant transfer of reducing equivalents from NADPH to molecular oxygen, thus diverting electrons from cytochrome P-450 enzymes. Metabolite separations showed a decrease in microsomal metabolism of benzo(a)pyrene and of benzo(a)pyrene-7,8-dihydrodoil upon addition of naphthazarin. Since both NADP and dicoumarol inhibited the naphthazarin-stimulated non-stoichiometric consumption of NADPH and oxygen then naphthazarin redox cycling probably involves both DT-diaphorase and NADPH cytochrome P-450 reductase.